We previously showed that radical metabolites of various xenobiotics and endogenous compounds can lead to the covalent bonding of the heme prosthetic group of hemeproteins to their apoproteins. In the case of myoglobin, the covalently altered protein has been shown to have increased reductive activity and rapidly reduced molecular oxygen to presumably the toxic metabolite superoxide anion radical. It was also shown by peptide mapping and mass spectrometry that the protein-bound adduct formed in the reaction of bromotrichloromethane with myoglobin consisted of a heme-CC12 moiety bound to the proximal histidine residue. This year we have obtained 1D and 2D proton NMR and C13 NMR spectra of this product and have identified for the first time the site of covalent attachment of a protein to its heme prosthetic group in a hemeprotein adduct. In this case, the proximal histidine residue was found to be covalently bonded to a vinyl substituent of the heme moiety. We have also investigated whether bromotrichloro- methane reacts similarly with other hemeproteins. Hemoglobin formed two heme adducts to its subunit. One of them was similar to that of myoglobin and contained a heme-CC12 moiety whereas the other had a covalently bound heme-CC13 moiety. Cytochrome P-450 and chloroperoxidase, but not horseradish peroxidase, formed covalently altered heme products when they were incubated with bromotrichloromethane. It is believed that the reaction of bromotrichloromethane with hemeproteins can be used to determine the amino acid residues at the active site of many hemeproteins by labeling these residues with activated heme moieties. Moreover, since the covalently altered hemeproteins may also have altered catalytic properties, it is possible that the metabolism-based activation of hemeproteins by radical metabolites of xenobiotics and endogenous compound may play a role in variety of tissue injuries.